Journal of Plant Growth Regulation

, Volume 24, Issue 1, pp 2–10

Efficient Method of Agrobacterium-mediated Transformation for Triticale (x Triticosecale Wittmack)

  • A. Nadolska-Orczyk
  • A. Przetakiewicz
  • K. Kopera
  • A. Binka
  • W. Orczyk
Article

Abstract

Transgenic plants of triticale cv. Wanad were obtained after transformation using three combinations of strain/vectors. Two of them were hypervirulent Agrobacterium tumefaciens strains (AGL1 and EHA101) with vectors containing bar under maize ubiquitin 1 promoter (pDM805), and both hpt under p35S and nptII under pnos (pGAH). The third one was a regular LBA4404 strain containing super-binary plasmid pTOK233 with selection genes the same as in pGAH. The efficiency of transformation was from 0 to 16% and it was dependent on the selection factor, auxin pretreatment, and the strain/vector combination. The highest number of transgenic plants was obtained after transformation with LBA4404(pTOK233) and kanamycin selection. Pretreatment of explants with picloram led to the highest number of plants obtained after transformation with both Agrobacterium/vector systems LBA4404(pTOK233) and EHA101(pGAH) and selected with kanamycin. Transgenic character of selected plants was examined by PCR using specific primers for bar, gus, nptII, and hpt and confirmed by Southern blot hybridization analysis. There was no GUS expression in T0 transgenic plants transformed with gus under p35S. However the GUS expression was detectable in the progeny of some lines. Only 30% of 46 transgenic lines showed Mendelian segregation of GUS expressing to GUS not expressing plants. In the remaining 70% the segregation was non-Mendelian and the rate was much lower than 3:1. Factors that might effect expression of transgenes in allohexaploid monocot species are discussed.

Keywords

Triticale Agrobacterium tumefaciens Cereal transformation Transgene expression PCR analysis 

References

  1. Barakat A, Gallois P, Raynal M, Maestre-Ortega D, Sallaud CH. 2000. The distribution of T-DNA in the genomes of transgenic Arabidopsis and rice. FEBS Lett 471:161–164CrossRefPubMedGoogle Scholar
  2. Becker D, Brettschneider R, Lörz H. 1994 Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant J 5:299–307CrossRefPubMedGoogle Scholar
  3. Chateau S, Sangwan RS, Sangwan-Norreel BS. 2000. Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones. J Exp Bot 51:1961–1968CrossRefPubMedGoogle Scholar
  4. Chen WP, Chen PD, Liu DJ, Kynast R, Friebe B. 1999 Development of wheat scab symptoms is delayed in transgenic wheat plants that constitutively express a rice thaumatin-like protein gene. Theor Appl Genet 99:755–760CrossRefGoogle Scholar
  5. Chen WP, Gu X, Liang GH, Muthukrishnan S, Chen PD. 1998. Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker. Theor Appl Genet 97:1296–1306CrossRefGoogle Scholar
  6. Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM. 1997 Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980PubMedGoogle Scholar
  7. Dai S, Zheng P, Marmey P, Zhang S, Tian W, 2001 Comparative analysis of transgenic rice plants obtained by Agrobacterium –mediated transformation and particle bombardment. Mol Breed 7:25–33CrossRefGoogle Scholar
  8. De Neve M, De Buck S, Jacobs A, Van Montagu M, Depicker A. 1997. T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J 11:5–29CrossRefGoogle Scholar
  9. Hayashi H, Alia, Mustardy L, Deshnium P, Ida M, Murata N. 1997. Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J 12:133–142CrossRefPubMedGoogle Scholar
  10. Hiei Y, Ohta S, Komari T, Kumashiro T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282CrossRefPubMedGoogle Scholar
  11. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilprroot RH 1983. A binary plant vector strategy based on separation of vir and T-region of Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180CrossRefGoogle Scholar
  12. Hood EE, Helmer GL, Fraley RT, Chilton M-D. 1986. The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301PubMedGoogle Scholar
  13. Hu T, Metz S, Chay C, Zhou HP, Biest N. 2003. Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep 21:1010–1019CrossRefPubMedGoogle Scholar
  14. Jefferson RA, Burgess SM, Hirsh D. 1986. β-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83:8447–8451.PubMedGoogle Scholar
  15. Jefferson RA, Kavanagh A, Bevan MW. 1987. GUS fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  16. Khanna HK, Daggard GE. 2003. Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium. Plant Cell Rep 21:429–436PubMedGoogle Scholar
  17. Kohli A, Leech M, Vain P, Laurie DA, Christou P. 1998. Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proc Natl Acad Sci USA 95:7203–7208CrossRefPubMedGoogle Scholar
  18. Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T. 1996. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174CrossRefPubMedGoogle Scholar
  19. Kooter JM, Matzke MA, Meyer P. 1999. Listening to the silent genes: transgene silencing, gene regulation and pathogen control. Trends in Plant Sci 4:340–347CrossRefGoogle Scholar
  20. Lazo GR, Stein PA, Ludwig RA. 1991. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Bio/Technology 9:963–967CrossRefPubMedGoogle Scholar
  21. Matzke MA, Matzke AJM. 1995. How and why do plants inactivate homologous (trans) genes. Plant Physiol 107:679–685PubMedGoogle Scholar
  22. Moore G. 2000 Cereal chromosome structure, evolution and pairing. Annu Rev Plant Physiol Plant Mol Biol 51:195–222 CrossRefPubMedGoogle Scholar
  23. Nadolska-Orczyk A, Orczyk W. 2003. Agrobacterium-mediated transformation of cereals. In: Jaiwal PK, Singh RP (eds.) Plant genetic engineering, vol 2. Improvement of food crops. Sci Tech Publishing LLC USA, pp 1–25.Google Scholar
  24. Nadolska-Orczyk A, Orczyk W, Przetakiewicz A. (2000) Agrobacterium-mediated transformation of cereals—from technique development to its application. Acta Physiol Plant 22:77–88Google Scholar
  25. Onouchi H, Yokoi K, Machida C, Matsuzaki H, Oshima Y. 1991. Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. Nucleic Acids Res 19:6373–6378PubMedGoogle Scholar
  26. Osborn TC, Pires JCH, Birchler JA, Auger DL, Chen ZJ (2003) understanding mechanisms of novel gene expression in polyploids. Trends Genetics 19:141–147CrossRefGoogle Scholar
  27. Pawlowski WP, Torbert KA, Rines HW, Somers DA. 1998. Irregular patterns of transgene silencing in allohexaploid oat. Plant Mol Biol 38:597–607CrossRefPubMedGoogle Scholar
  28. Przetakiewicz A, Karas A, Orczyk W, Nadolska-Orczyk A. 2004. Agrobacterium-mediated transformation of polyploid cereals. The efficiency of selection and transgene expression in wheat. Cell Mol Biol Lett 9: 903–917Google Scholar
  29. Przetakiewicz A, Orczyk W, Nadolska-Orczyk A. 2003. The effect of auxin on plant regeneration of wheat, barley and triticale. Plant Cell Tissue Org Cult 73:245–256CrossRefGoogle Scholar
  30. Sangwan RS, Bourgeois Y, Brown S, Vasseur G, Sangwan-Norreel BS. 1992. Characterisation of competent cells and early events of Agrobacterium-mediated genetic transfromation in Arabidopsis thaliana. Planta 188:439–456CrossRefGoogle Scholar
  31. Stoger E, Williams S, Keen D, Christou P 1999. Constitutive versus seed specific expression in transgenic wheat: temporal and spatial control. Transgenic Res 8:73–82CrossRefGoogle Scholar
  32. Svitashev S, Ananiev E, Pawlowski WP, Somers DA. 2000. Association of transgene integration sites with chromosome rearrangements in hexaploid oat. Theor Appl Genet 100:872–880CrossRefGoogle Scholar
  33. Tingay S, McElroy D, Kalla R, Fieg S, Wang M. 1997. Agrobacterium tumefaciens-mediated barley transformation. Plant J 11:1369–1376CrossRefGoogle Scholar
  34. Villemont E, Dubois F, Sangwan RS, Vasseur G, Bourgeois Y. (1997). Role of the host cell cycle in Agrobacterium-mediated genetic transformation of Petunia: evidence of S-phase control mechanism for T-DNA transfer. Planta 201:160–172CrossRefGoogle Scholar
  35. Wu H, Sparks C, Amoah B, Jones HD. 2003. Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep 21:659–668PubMedGoogle Scholar
  36. Zhao Z, Gu W, Cai T, Tagliani L, Hondred D. 001. High throughout genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed 5:323–333Google Scholar
  37. Zhou H, Arrowsmith JW, Fromm ME, Hironaka CM, Taylor ML. 1995. Glyphosate tolerant CP4 and GOX genes as a selectable marker in wheat transformation. Plant Cell Rep 15: 59–163CrossRefGoogle Scholar
  38. Zimny J, Becker D, Brettschneider R, Lörz H (1995) Fertile, transgenic Triticale (xTriticosecale Wittmack). Mol Breed 1:155–164CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • A. Nadolska-Orczyk
    • 1
  • A. Przetakiewicz
    • 1
  • K. Kopera
    • 1
  • A. Binka
    • 1
  • W. Orczyk
    • 1
  1. 1.Plant Transformation and Cell Engineering LabPlant Breeding and Acclimatization InstituteRadzikowPoland

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